Functional study of acetylcholinesterase genes in Bombyx mori ovary cells using RNA interference

Abstract

Acetylcholinesterase (AChE) plays a key role in neurotransmission, in which it maintains the transmission of nerve impulses by catalyzing acetylcholine in the neuronal synapse. Acetylcholinesterase in Lepidoptera is encoded by two ace genes, ace1 and ace2. In this study, we used RNA interference to individually knock down the expression of ace1 or ace2 in a Bombyx mori (L.) (Lepidoptera: Bombycidae) cell line (BmN) to explore their functions and relationships. Knockdown efficiency was confirmed using quantitative real‐time PCR. We also monitored transcription levels of ace1 and ace2 at different time points after B. mori nucleopolyhedrovirus (BmNPV) infection. We found that when the expression of ace1 was knocked down to 37.5%, the expression of ace2 increased by 66%; when ace2 expression was reduced to 33.4%, the ace1 expression was similarly reduced to 26.7%. Acetylcholinesterase activity was decreased to 86.2% when ace2 expression was inhibited. However, no significant difference was observed in AChE activity when ace1 expression was knocked down. After BmNPV infection, ace1 expression was gradually increasing, whereas ace2 expression was gradually decreasing. Our study indicates that ace2 plays an important compensatory role for the loss of ace1 in maintaining AChE activity by increasing its expression level. In addition, ace2 may also regulate transcription of ace1. Overall, our findings help elucidate the relationship between the two ace genes in Lepidoptera.

RNA interference: Applications and advances in insect toxicology and insect pest management

“…Although the ability of AChE2 to function as a major catalytic enzyme does not necessarily indicate either its major function in cholinergic neurotransmission or as a major target for OP and CB insecticides, it would be interesting to investigate if AChE2 is a major enzyme responsible for cholinergic neurotransmission and serves as a major target for these insecticides by RNAi. Some studies have demonstrated RNAi-mediated silencing of ace genes in Chilo suppressalis [16], Plutella xylostella [12], Helicoverpa armigera [13] and Bombyx mori ovary cell lines [38]. Although changes of the insecticide susceptibility in these insects were not evaluated after RNAi, these studies suggest that RNAi can be used to determine the target gene for OP and CB insecticides in a wide range of insect species possessing both ace1 and ace2 genes.…”

“…These findings strongly support that AChE1 plays a major role in cholinergic functions, and is the major target of anticholinesterase insecticides. However, more recent studies revealed that some insects express AChE2 as the major catalytic enzyme, rather than AChE1 [28,37,38]. Although the ability of AChE2 to function as a major catalytic enzyme does not necessarily indicate either its major function in cholinergic neurotransmission or as a major target for OP and CB insecticides, it would be interesting to investigate if AChE2 is a major enzyme responsible for cholinergic neurotransmission and serves as a major target for these insecticides by RNAi.…”

EXPRESSION AND EFFECTS OF MUTANT Bombyx mori ACETYLCHOLINESTRASE1 IN BmN CELLS

“…Cao et al. () found that the expression of ace 2 was significantly increased (by 66%) when the expression of ace 1 was knocked down to 37.4% of its normal level. In this study, we found that ace 2 expression was significantly reduced when ace 1's level was increased by transfection.…”

Expression and functional analysis of ace1 and ace2 reveal their differential roles in larval growth and insecticide sensitivity in Spodoptera frugiperda (J. E. Smith, 1797)

“…In non-Cyclorrhapha insects, ace1 has been proposed as the major catalytic enzyme in acetylcholine hydrolyzation in insects (Kim and Lee 2013) based on its higher expression level than ace2 in Plutella xylostella (Baek et al 2005), Helicoverpa assulta (Lee et al 2006), Aedes albopictus (Mizuno et al 2006), Tribolium castaneum (Lu et al 2012), Spodoptera exigua (Zhao et al 2019), Spodoptera litura (Salim et al 2017) and Blattella germanica (Revuelta et al 2009). However, in some insect species, such as Apis mellifera (Kim et al 2012b), Bombyx mori (Cao et al 2012;Chen et al 2009), the tissue distribution pattern, kinetic properties and catalytic e ciency of AChE2 are signi cantly higher than those of AChE1 (Kim et al 2012b).…”